To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.
Part 1: Transfection of HEK 293 Cells To Produce Lentiviruses
*24 hours before transfection, plate 2.5 X 106 cells in a 10cm dish for a confluency of 50-70% the next day.
Part 2: Lentiviral Harvest
Part 3: Lentiviral Concentration via Ultracentrifugation
Part 4: Lentiviral Titration Using Flow Cytometry
Calculate the number of infectious units per ml with the following formula:
Part 5: Representative Results:
After a 48-hour incubation period post transfection, the plate of 293 cells should be at around 90% fluorescent. A high percentage of fluorescent cells is an indication of a high percentage of virus producing cells.
After centrifugation, the viral pellet is barely visible when resuspending. This is normal as the pellet is clear and very small.
When interpreting flow cytometer results, note that one can only achieve accurate titer calculations when infected (fluorescent) cells have received no more than one viral integration per cell. In order to ensure that this is the case, use cells with < 15 % infection rate for calculations. Cells with higher infection rates have likely received multiple viral integrants per cell. The dilutions suggested in step 2 should yield several samples within this range of infection. Ideally, use several samples to generate a titration plot (linear line) from which you can calculate the final titer, but you can calculate titer using a single sample, if necessary. One can expect titer of 1.0 x 107 TU/ml for un-concentrated virus and 1.0 x 108 TU/ml for concentrated virus.
Sandler Asthma Basic Research Center (SABRE)
Sandler Foundation
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
DMEM HEK 293T cells Penicillin-streptomycin Glutamine Phosphate-buffered saline FBS | Invitrogen | |||
FuGENE 6 transfection reagent pSico mCherry pMDL pRSV pVSVG | Roche | |||
Bleach | Clorox | |||
10-cm cell culture dishes 96 well cell culture dishes | Falcon | 353003 | ||
Multichannel pipette | Rainin | |||
Filtered pipette tips | Rainin | |||
1.5ml Eppendorf tubes Syringe Syinge filter (0.45-μm) | Eppendorf | |||
Ultracentrifuge tubes | Beckman | |||
Tissue culture incubator at 5% CO2 | Coulter | |||
Beckman SW41T.I rotor | ||||
Beckman ultracentrifuge | Beckman | |||
Fluorescent microscope | Coulter | |||
FACS Array | Beckman |